Interferon production in microcarrier culture of human fibroblast cells.

The microcarrier cell culture technique offers a convenient way of manipulating relatively large amounts of anchorage-dependent cultured mammalian cells in a relatively small volume. Other advantages of this system are ease of direct observation, and of monitoring and control of other culture parameters. The production of human fibroblasts interferon on microcarrier culture on a scale of up to 44 litres (very roughly equivalent to 1100 roller bottles) using a conventional inducation with poly(I) poly(C), followed by superinduction by treatment with metalolic inhibitors, is described.

Skin reactions to interferon inoculations are reduced but not abolished by purification.

The skin reactions to partially-purified fibroblast-derived (HulFN-beta) and leucocyte-derived (HulFN-alpha) interferons and to HulFN-alpha of increasing purity were studied in normal volunteers. Reactions consisted of an immediate flare followed by a dense well-circumscribed erythema maximal at 4-8 hours. HulFN-beta caused significantly larger reactions than HulFN-alpha of approximately the same purity. Immediate flare reactions occurred even to the most pure preparation tested but the late reactions were significantly reduced. Experiments suggested that the early reactions to impure HulFN-alpha were in part mediated by local histamine release and late reactions in part by local prostaglandin synthesis. This suggests that interferon itself may not be the cause of general symptoms during a virus illness.

Mechanism of binding of mouse interferon to controlled pore glass.

Many proteins bind to controlled pore glass; they are either acid elutable or alkali elutable. Mouse interferon is an acid-elutable protein. Since poly(L-lysine) and, to some extent, poly(L-arginine) are also eluted from controlled pore glass under acidic conditions, one may postulate that mouse interferon binds to controlled pore glass via some of the protein's epsilon-amino groups (of lysine) and/or guanidinium groups (of arginine) and the beads' silanol (hydroxyl groups). The necessity of lysine in the binding of interferon to controlled pore glass is further substantiated by the fact that citraconylated interferon does not bind to controlled pore glass. A requirement for Lewis acid-base interaction between the beads' B2O3 groups and the amide groups of arginine is unlikely in view of the results obtained with the alternative system, ZrOH, which, being devoid of B2O3, did bind interferon. Since a substantial amount of interferon could be eluted from controlled pore glass with ethylene glycol and high salt, one may assume that some hydrophobicity is involved in the binding of interferon to controlled pore glass.

Human fibroblast interferon for clinical trials: production, partial purification, and characterization.

The production and partial purification of human fibroblast interferon for performing clinical trials is described. The interferon was produced by superinduction (exposure to riboinosinic-ribocytidylic acid, cycloheximide, and actinomycin D) of large numbers of human diploid fibroblast cultures. The yield averaged 750 units per cm(2) of culture area. The interferon was concentrated and purified by a two-step procedure involving acid desorption from controlled-pore glass beads and dialysis against polyethylene glycol. Human plasma protein was added as a stabilizer. The lyophilized end product had a specific activity of 0.5 x 10(6) to 1 x 10(6) units/mg of protein; it could be reconstituted for injection at a concentration of 2 x 10(6) units/ml. The composition of this interferon was characterized by crossed immunoelectrophoresis with polyspecific antibodies prepared against the principal sources of potential contaminants: human serum, calf serum, and normal human fibroblasts. Several components of each source were detected. Although the major component of calf serum, bovine serum albumin, was absent, other minor components were retained by the production and purification sequence. One of the main contaminants of fibroblast origin was found to be fibronectin.

Human fibroblast interferon for clinical trials: pharmacokinetics and tolerability in experimental animals and humans.

Human fibroblast interferon (F-interferon) purified by adsorption on controlled-pore glass was given intramuscularly to patients at daily dosages of up to 20 x 10(6) units. Serum levels of antiviral activity were low or undetectable. In contrast, reasonably high serum titers were found in patients receiving interferon prepared from leukocytes (L-interferon). Similarly, in rabbits lower serum titers were seen with F-interferon than with L-interferon. These results are at variance with those obtained earlier (V. G. Edy, A. Billiau, and P. De Somer, J. Infect. Dis. 133:A18-A21, 1976). Possible explanations for this discrepancy are discussed. The F-interferon evoked febrile reactions, delayed skin reactivity, and transitory lymphopenia in humans. Some patients developed an allergic state of the reaginic type as evidenced by a weal and flare reaction after intradermal challenge. However, these patients did not show allergic symptoms after intramuscular injections. None of the side effects was severe enough to prohibit continuation of the treatment; most of them seemed to be due to contaminants not removed by the purification method. The possibility is considered that some of the side effects, e.g., delayed skin reactivity, are sufficiently specific to justify identification of the active principals.

Double-blind study of interferon administration in renal transplant recipients.

In a double-blind trial renal allograft recipients were treated with fibroblast interferon preparations for 3 months in an attempt to prevent viral infections. Interferon therapy did not reduce the overall incidence of viral infections. No adverse effects were noted on liver function, platelet counts, or leucocyte counts, or acute rejection episodes.

Purification of human fibroblast interferon by zinc chelate affinity chromatography.

Zinc chelated by iminodiacetic acid linked to an insoluble matrix binds human fibroblast interferon quite selectively at neutral pH in 0.15 M NaCL. On reduction of the pH and increase of the ionic strength, the interferon is eluted. Using a pH gradient at constant high ionic strength, good purification of the interferon can be obtained, up to a final specific activity of 108.5 units/mg of protein. Approximately 60% of the applied activity can be recovered.

Human interferon: mass production in a newly established cell line, MG-63.

MG-63 cells, a line derived from an osteosarcoma, produced high yields of interferon after superinduction with polyinosinic acid.polycytidylic acid, cycloheximide, and actinomycin D. Advantages of MG-63 cells over diploid fibroblasts as a substrate are: no requirement for aging between confluency and induction, no requirement for priming, and 3.7-fold higher yields per square centimeter of culture surface. Physicochemically and biologically, MG-63 cell interferon resembles fibroblast rather than leukocyte interferon.

Stable and unstable forms of human fibroblast interferon.

There is a minor fraction of human fibroblast interferon that resembles human leukocyte interferon in being renaturable after treatment with guanidine hydrochloride. However, antigenically and in its low activity on heterologous cells, it resembles the bulk of human fibroblast interferon. Since the production of this stable interferon fraction is not greatly inhibited by glucosamine at concentrations that significantly reduce total interferon production, it is suggested that it differs from the bulk of human fibroblast interferon in the extent or nature of glycosylation.

Purification of interferon by adsorption chromatography on controlled pore glass.

Human fibroblast and mouse L929 cell interferons can be purified by adsoprtion to and subsequent elution from Controlled Pore Glass. Purification of 40 to 90-fold to specific activities of 1 to 5 times 10(6) units/mg of protein can be achieved in a single step, with good recovery of activity. Human leukocyte interferon does not bind to the glass and cannot be purified in this way.